RESUMO
Meiotic recombination, crucial for proper chromosome segregation and genome evolution, is initiated by programmed DNA double-strand breaks (DSBs) in yeasts and likely all sexually reproducing species. In fission yeast, DSBs occur up to hundreds of times more frequently at special sites, called hot spots, than in other regions of the genome. What distinguishes hot spots from cold regions is an unsolved problem, although transcription factors determine some hot spots. We report the discovery that three coiled-coil proteins-Rec25, Rec27, and Mug20-bind essentially all hot spots with great specificity even without DSB formation. These small proteins are components of linear elements, are related to synaptonemal complex proteins, and are essential for nearly all DSBs at most hot spots. Our results indicate these hot spot determinants activate or stabilize the DSB-forming protein Rec12 (Spo11 homolog) rather than promote its binding to hot spots. We propose a paradigm for hot spot determination and crossover control by linear element proteins.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Meiose , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Recombinação Genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The endothelial protein C receptor (EPCR) plays an important role in cardiovascular disease by binding protein C/activated protein C (APC). EPCR structure contains a hydrophobic groove filled with an unknown phospholipid needed to perform its function. It has not been established whether lipid exchange takes place in EPCR as a regulatory mechanism of its activity. Our objective was to identify this phospholipid and to explore the possibility of lipid exchange as a regulatory mechanism of EPCR activity driven by the endothelially expressed secretory group V phospholipase A(2) (sPLA(2)-V). We identified phosphatidylcholine (PCh) as the major phospholipid bound to human soluble EPCR (sEPCR). PCh in EPCR could be exchanged for lysophosphatidylcholine (lysoPCh) and platelet activating factor (PAF). Remarkably, lysoPCh and PAF impaired the protein C binding ability of sEPCR. Inhibition of sPLA(2)-V, responsible for lysoPCh and PAF generation, improved APC binding to endothelial cells. EPCR-dependent protein C activation and APC antiapoptotic effect were thus significantly enhanced. In contrast, endothelial cell supplementation with sPLA(2)-V inhibited both APC generation and its antiapoptotic effects. We conclude that APC generation and function can be modulated by changes in phospholipid occupancy of its endothelial cell receptor.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Fosfolipases A2 do Grupo V/metabolismo , Lisofosfatidilcolinas/química , Fator de Ativação de Plaquetas/química , Proteína C/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Animais , Cromatografia em Camada Fina , Células Endoteliais/metabolismo , Receptor de Proteína C Endotelial , Ativação Enzimática/fisiologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Lisofosfatidilcolinas/metabolismo , Espectrometria de Massas , Camundongos , Fator de Ativação de Plaquetas/metabolismo , Estrutura Terciária de Proteína , Espectrometria de FluorescênciaRESUMO
BACKGROUND: To our knowledge, there is scant literature on comparative broiler response to cereal diets high in soluble non-starch polysaccharides without or with enzyme, prebiotic, probiotic or synbiotic supplementation. In the present study, the effects of a wheat- and barley-based diet with or without supplemental xylanase plus ß-glucanase, inulin, Enterococcus faecium or inulin plus Enterococcus faecium, on bird performance, digesta viscosity, nutrient digestibility and intestinal microflora were compared to a maize-based diet. RESULTS: In comparison to a maize-based diet, the wheat- and barley-based diet reduced (P < 0.05) body weight gain and feed intake, but did not affect to the feed-to-gain ratio. Apparent digestibility of crude fat and various fatty acids were decreased (P < 0.05) as well as apparent metabolisable energy corrected to zero nitrogen retention content. There was an increase (P < 0.05) in the viscosity of jejunal digesta and in the caecal numbers of Escherichia coli and lactobacilli, and a decrease in the ileal numbers of E. coli and lactobacilli. Performance parameters and nutrient digestibility were not affected (P > 0.05) by dietary inclusion of the additives used, with the exception that exogenous enzyme improved (P < 0.05) the apparent digestibility of crude fat and decreased the viscosity of jejunal digesta. Enzyme and Enterococcus faecium supplementation increased intestinal lactic acid bacteria, whereas inulin addition reduced the number of E. coli (P < 0.05). Addition of inulin-Enterococcus faecium decreased E. coli and increased bifidobacteria numbers in the caeca. CONCLUSION: Enzyme supplementation to a wheat- and barley-based diet significantly improved the apparent digestibility of dietary fat. All four additives had a beneficial effect on the intestinal microflora of broilers.
Assuntos
Ração Animal , Galinhas , Dieta , Suplementos Nutricionais , Digestão , Enzimas/farmacologia , Inulina/farmacologia , Probióticos/farmacologia , Animais , Bifidobacterium/crescimento & desenvolvimento , Ceco/microbiologia , Celulases , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Galinhas/microbiologia , Ingestão de Energia , Metabolismo Energético , Enterococcus faecium , Escherichia coli/crescimento & desenvolvimento , Gorduras/metabolismo , Ácidos Graxos/metabolismo , Aditivos Alimentares/farmacologia , Conteúdo Gastrointestinal , Hordeum , Íleo/microbiologia , Intestinos/microbiologia , Jejuno , Lactobacillus/crescimento & desenvolvimento , Carne , Nitrogênio/metabolismo , Prebióticos , Triticum , Viscosidade , Aumento de Peso , Xilosidases , Zea maysRESUMO
The objectives of the current study were (1) to evaluate the effect of sprouting on protein, amino acids, fats, fatty acids, starch, total soluble carbohydrates, and ß-D-glucan content of barley grains and (2) to know the content of these nutrients in the morphological fractions of sprouts: green shoot, residual structure of sprouted grain (RSSG), residual structure of sprouted grain plus unsprouted grain (RSSG plus UG), and root fractions and to determine the proportion of each of these fractions (on fresh and dry basis) in the sprout biomass. Barley grain was sprouted in a commercial germination chamber for a period of 6 days. Raw grain was used as a control. Results showed that crude protein, ether extract, total soluble carbohydrates, and cellulose content increased, whereas starch and ß-D-glucan content decreased in sprouted when compared with the control grain. Amino acid and fatty acid profiles were also affected. Thus, aspartic acid, threonine, alanine, valine, isoleucine, lysine, and tryptophan content increased and only that of glutamic acid decreased after sprouting. Regarding fatty acids, an increase in the relative concentration of C18 : 0 and C18:3n-3 and a decrease in that of C18:1n-9 were detected. Partitioning of sprouted barley into three morphological component fractions showed that the residual structures of sprouted grains plus unsprouted grain fraction made up 82.9% and 93.6% of sprout biomass, on fresh and DM basis, respectively, and the remainder was provided by the root fraction, 10.3% and 3.2%, respectively, and by the green shoot fraction, 6.8% and 3.1%, respectively. The three morphological fractions differed in the content of the most analyzed nutrients.
RESUMO
Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csb(m/m)/Xpa(-/-) mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csb(m/m)/Xpa(-/-) mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csb(m/m)/Xpa(-/-) and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair-deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.
Assuntos
Síndrome de Cockayne/genética , Reparo do DNA , Genoma/genética , Hormônio do Crescimento/genética , Fator de Crescimento Insulin-Like I/metabolismo , Envelhecimento , Animais , Antioxidantes/farmacologia , Síndrome de Cockayne/etiologia , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Dietilexilftalato/farmacologia , Ácidos Graxos/biossíntese , Glucose/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Poli-ADP-Ribose , Radiação Ionizante , Somatotrofos/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
Inclusion of prebiotics in the diet is known to be advantageous, with positive influences both on health and growth. The current study investigated the differences in the hepatic transcriptome profiles between chickens supplemented with inulin (a storage carbohydrate found in many plants) and controls. Liver is a major metabolic organ and has been previously reported to be involved in the modification of the lipid metabolism in chickens fed with inulin. A nutrigenomic approach through the analysis of liver RNA hybridized to the Affymetrix GeneChip Chicken Genome Array identified 148 differentially expressed genes among both groups: 104 up-regulated (≥ 1.4-fold) and 44 down-regulated (≤ 0.6-fold). Quantitative real-time PCR analysis validated the microarray expression results for five out of seven genes tested. The functional annotation analyses revealed a number of genes, processes and pathways with putative involvement in chicken growth and performance, while reinforcing the immune status of animals, and fostering the production of long chain fatty acids in broilers supplemented with 5 g of inulin kg(-1) diet. As far as we are aware, this is the first report of a microarray based gene expression study on the effect of dietary inulin supplementation, supporting further research on the use of this prebiotic on chicken diets as a useful alternative to antibiotics for improving performance and general immunity in poultry farming, along with a healthier meat lipid profile.
Assuntos
Galinhas/genética , Suplementos Nutricionais , Regulação da Expressão Gênica , Inulina/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Transcriptoma , Análise de Variância , Animais , Análise por Conglomerados , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Inulina/farmacologia , Anotação de Sequência Molecular , Prebióticos , Reprodutibilidade dos TestesAssuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Glicólise/genética , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Oncogenes , Fosforilação Oxidativa , Serina/metabolismoRESUMO
Yeast Sir2 deacetylase is a component of the silent information regulator (SIR) complex encompassing Sir2/Sir3/Sir4. Sir2 is recruited to telomeres through Rap1, and this complex spreads into subtelomeric DNA via histone deacetylation. However, potential functions at telomeres for SIRT1, the mammalian orthologue of yeast Sir2, are less clear. We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1(super)) mouse models. Our results indicate that SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging, an effect dependent on telomerase activity. Using chromatin immunoprecipitation assays, we find that SIRT1 interacts with telomeric repeats in vivo. In addition, SIRT1 overexpression increases homologous recombination throughout the entire genome, including telomeres, centromeres, and chromosome arms. These findings link SIRT1 to telomere biology and global DNA repair and provide new mechanistic explanations for the known functions of SIRT1 in protection from DNA damage and some age-associated pathologies.
Assuntos
Recombinação Genética/genética , Sirtuína 1/genética , Telômero/metabolismo , Acetilação , Envelhecimento/genética , Animais , Centrômero/genética , Centrômero/metabolismo , Fragilidade Cromossômica/genética , Cromossomos/genética , Cromossomos/metabolismo , Dano ao DNA/genética , Metilação de DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Expressão Gênica/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/genética , RNA/genética , Troca de Cromátide Irmã/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genéticaRESUMO
Some physicochemical and rheological properties of the exopolysaccharide (EPS) produced by Pediococcus parvulus 2.6 were examined. Structural characterization by NMR ((1)H and 2D-COSY) showed that the same EPS, a 2-substituted (1,3)-beta-D-glucan, was synthesized irrespective of sugar source used for growth (glucose, fructose, or maltose). The molecular masses of these beta-glucans were always very high (>10(6) Da) and influenced by the culture medium or sugar source. The steady shear rheological experiments showed that all concentrations of the beta-glucan aqueous solutions exhibited a pseudoplastic behavior at high shear rates. Viscoelastic behavior of beta-glucan solutions was determined by dynamic oscillatory analysis. A critical concentration of 0.35% associated with the appearance of entanglements was calculated. The beta-glucan adopts an ordered hydrogen bond dependent helical conformation in neutral and slightly alkaline aqueous solutions, which was partly denatured under more alkaline conditions.
Assuntos
Pediococcus/química , beta-Glucanas/química , Estrutura Molecular , Peso Molecular , Pediococcus/metabolismo , Reologia , beta-Glucanas/metabolismoRESUMO
Mice defective for the Polk gene, which encodes DNA polymerase kappa, are viable and do not manifest obvious phenotypes. The present studies document a spontaneous mutator phenotype in Polk(-/-) mice. The initial indication of enhanced spontaneous mutations in these mice came from the serendipitous observation of a postulated founder mutation that manifested in multiple disease states among a cohort of mice comprising all three possible Polk genotypes. Polk(-/-) and isogenic wild-type controls carrying a reporter transgene (the lambda-phage cII gene) were used for subsequent quantitative and qualitative studies on mutagenesis in various tissues. We observed significantly increased mutation frequencies in the kidney, liver, and lung of Polk(-/-) mice, but not in the spleen or testis. G:C base pairs dominated the mutation spectra of the kidney, liver, and lung. These results are consistent with the notion that Pol kappa is required for accurate translesion DNA synthesis past naturally occurring polycyclic guanine adducts, possibly generated by cholesterol and/or its metabolites.
Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Mutação , Fenótipo , Animais , Pareamento de Bases , Linhagem Celular , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/genética , Genes Reporter , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: The diagnosis of inflammatory bowel disease is supported by clinical findings and complementary tests. The presence of specific serological markers could be helpful in the characterization of this condition. AIM: To assess the prevalence of ANCA and ASCA in a group of patients with ulcerative colitis (UC) and its association with clinical features. MATERIAL AND METHODS: Sixty four patients with UC in remission (age range 16-72 years, 33 males) were studied. In a venous blood sample ANCA were measured by indirect immunofluorescence and ASCA by enzyme immune assays for IgG and IgA. RESULTS: Forty four percent of patients were positive for ANCA, 9% for ASCA and 6% for both markers. There was a significant correlation between the presence of ANCA and duration of the UC (<5 years 50%, 5-10 years 42.9%, 15 years 30%) and the number of crises (one crises 31%, 2-5 crises 51.9% and >5 crises 87.5). The proportion of colectomized patients with positive ANCA was higher (57.1%). CONCLUSIONS: The prevalence of ANCA in the studied population is similar to the published data. The presence of ANCA was significantly higher in UC patients with shorter evolution, higher number of crises and in those with a history of colectomy. There was a low prevalence of ASCA positive patients.